Contribution of C-tail residues of potato carboxypeptidase inhibitor to the binding to carboxypeptidase A: A mutagenesis analysis

Cristina Marino-Buslje, Gabriela Venhudová, Miguel A. Molina, Baldomero Oliva, Xavier Jorba, Francesc Canals, Francesc X. Avilés, Enrique Querol

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18 Citations (Scopus)

Abstract

The role of each residue of the potato carboxypeptidase inhibitor (PCI) C-terminal tail, in the interaction with carboxypeptidase A (CPA), has been studied by the analysis of two main kinds of site-directed mutants: the point substitution of each C-terminal residue by glycine and the sequential deletions of the C-terminal residues. The mutant PCI-CPA interactions have been characterized by the measurement of their inhibition constant, K(i), in several cases, by their kinetic association and dissociation constants determined by presteady-state analysis, and by computational approaches. The role of Pro36 appears to be mainly the restriction of the mobility of the PCI C-tail. In addition, and unexpectedly, both Gly35 and Pro36 have been found to be important for folding of the protein core. Val38 has the greatest enthalpic contribution to the PCI-CPA interaction. Although Tyr37 has a minor contribution to the binding energy of the whole inhibitor, it has been found to be essential for the interaction with the enzyme following the cleavage of the C-terminal Gly39 by CPA. The energetic contribution of the PCI secondary binding site has been evaluated to be about half of the total free energy of dissociation of the PCI-CPA complex.
Original languageEnglish
Pages (from-to)1502-1509
JournalEuropean Journal of Biochemistry
Volume267
Issue number5
DOIs
Publication statusPublished - 22 Mar 2000

Keywords

  • Carboxypeptidase
  • Enzyme-inhibitor complex
  • Inhibition kinetics
  • Mutational analysis
  • Potato carboxypeptidase inhibitor

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